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Methods of Measuring Internalization of G Protein–Coupled Receptors

Nicholas Evans1

1GlaxoSmithKline Research, Harlow, United Kingdom

Publication Name: 
Current Protocols in Pharmacology
Unit Number: 
Unit 12.6
DOI: 
10.1002/0471141755.ph1206s24
Online Posting Date: 
May, 2004
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Abstract

This unit provides detailed protocols for measuring receptor internalization. The techniques are sufficiently generalized to be applicable to most receptors in a wide variety of cell types. Both radioactive and non-radioactive techniques are described that may be used to quantify receptor internalization, and the differences between the two are highlighted. This unit discusses how quantification of internalization may be achieved, and the advantages and drawbacks of each technique. Low- and higher-throughput methods are compared, and the technologies required to conduct the analyses are discussed.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Measurement of Receptor Internalization Using Radiolabeled Ligands
  • Alternate Protocol 1: Direct Measurement of Receptor Internalization after Radioligand Binding
  • Alternate Protocol 2: Separation of External and Internalized ligand Using Sucrose Density Centrifugation
  • Basic Protocol 2: Colorimetric Technologies to Measure Receptor Internalization Using Elisa
  • Alternate Protocol 3: Fluorescence Technologies to Measure Receptor Internalization Using Flow Cytometry
  • Basic Protocol 3: Analysis of Receptor Internalization in Fixed Cells Using Fluorescence Microscopy
  • Alternate Protocol 4: Monitoring Real-Time Receptor Internalization Using Time Lapse Confocal Microscopy
  • Alternate Protocol 5: Measurement of Internalization of Receptor–Green Fluorescent Protein Chimeras by Confocal Microscopy
  • Basic Protocol 4: Measuring Receptor Internalization Using High-Throughput Technologies
  • Support Protocol 1: Preparation of Cells for Receptor Internalization Studies
  • Support Protocol 2: Preparation of Ligand and Compound Plates for Receptor Internalization Studies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Receptor Internalization Using Radiolabeled Ligands

 Materials
  • Cells of interest seeded into poly-l-lysine-coated 24-well plates at 2 × 105 cells/well or 96-well plates at 1.5–2.5 × 104 cells/well (see Support Protocol 1)
  • Ligand of interest
  • HBSS/Ca2+ (see recipe), prewarmed to 37°C
  • PBS (see recipe), ice cold
  • Low-pH stripping buffer 1 (see recipe) or high-pH stripping buffer (see recipe), ice cold
  • Radioligand of interest
  • 0.1 M NaOH

Alternate Protocol 1: Direct Measurement of Receptor Internalization after Radioligand Binding

 Additional Materials (also see Basic Protocol 1)
  • Binding buffer (see recipe), 37°C and ice-cold
  • Low-pH stripping buffer 2 (see recipe), ice-cold
  • 0.5 M NaOH

Alternate Protocol 2: Separation of External and Internalized ligand Using Sucrose Density Centrifugation

 Additional Materials (also see Basic Protocol 1)
  • Serum-free EMEM (see recipe), prewarmed to 37°C
  • 1% (w/v) BSA in PBS (see recipe), ice-cold
  • Lysis buffer (see recipe)

Basic Protocol 2: Colorimetric Technologies to Measure Receptor Internalization Using Elisa

 Materials
  • Agonist for receptor of interest in HBSS/Ca2+ (see recipe)
  • Cells of interest in 24-well plates (~2 × 105 cells/well) coated with 0.1 mg/ml poly-l-lysine (see Support Protocol 1)
  • TBS (see recipe)
  • 4% (w/v) paraformaldehyde in TBS
  • 1% (w/v) BSA in TBS
  • Primary antibody for receptor of interest diluted in 1% (w/v) BSA in TBS
  • Alkaline phosphatase–conjugated secondary antibody, diluted according to manufacturer's instructions in 1% (w/v) BSA in TBS
  • Alkaline phosphatase substrate (e.g., Bio-Rad)
  • 96-well plate with 100 µl of 0.4 M NaOH in each well
  • ELISA plate reader (e.g., Spectramax; Molecular Devices)

Alternate Protocol 3: Fluorescence Technologies to Measure Receptor Internalization Using Flow Cytometry

 Additional Materials (also see Basic Protocol 2)
  • PBS (see recipe), ice cold and room temperature
  • Appropriate anti-epitope primary antibody diluted in 1% (w/v) BSA in PBS
  • Fluorescently tagged secondary antibody (e.g., 1:200 goat anti-mouse; Sigma) diluted in 1% (w/v) BSA in PBS
  • 10 mM Tris·Cl/5 mM EDTA, pH 7.4
  • 3.6% (w/v) paraformaldehyde in PBS
  • Additional reagents and equipment for flow cytometry

Basic Protocol 3: Analysis of Receptor Internalization in Fixed Cells Using Fluorescence Microscopy

 Materials
  • Cells of interest plated on glass coverslips in 6-well plates (see Support Protocol 1)
  • Appropriate growth medium (e.g., DMEM/30 mM HEPES, pH 7.4), 4°C
  • Fluorescently tagged transferrin (Molecular Probes) diluted in recommended buffer according to manufacturer's instructions
  • Appropriate anti-epitope primary antibody diluted in 1% (w/v) BSA in PBS (see recipe)
  • Appropriate agonist diluted in HBSS/Ca2+ (see recipe)
  • 3.7% (w/v) formaldehyde in PBS, pH 7.4 (see recipe)
  • 1 mM CaCl2 in TBS (see recipe)
  • Permeabilizing buffer (see recipe)
  • Appropriate FITC-labeled secondary antibody, diluted in 1% (w/v) BSA in PBS according to manufacturer's instructions
  • Additional reagents and equipment for fluorescence microscopy

Alternate Protocol 4: Monitoring Real-Time Receptor Internalization Using Time Lapse Confocal Microscopy

 Additional Materials (also see Basic Protocol 3)
  • Cells plated onto chambered coverglass slides for confocal microscopy (see Support Protocol 1)
  • PBS (see recipe), ice-cold
  • Additional reagents and equipment for confocal microscopy, including temperature-controlled stage

Alternate Protocol 5: Measurement of Internalization of Receptor–Green Fluorescent Protein Chimeras by Confocal Microscopy

 Additional Materials (also see Basic Protocol 3)
  • Cells expressing receptor-GFP chimera of interest, grown on microscope slides in 6-well plates (see Support Protocol 1)
  • PBS (see recipe), optional
  • 10 µg/ml red fluorescent lectin (e.g., rhodamine-concanavalin A; Molecular Probes) diluted in recommended buffer according to manufacturer's instructions, optional
  • Appropriate ligand
  • Software for image analysis (e.g., Zeiss LSM; Zeiss) and data analysis (e.g., GraFit, Erithacus Software or Prism, GraphPad Software)
  • Additional reagents and equipment for confocal or fluorescence microscopy, including temperature-controlled stage (for time course studies)

Basic Protocol 4: Measuring Receptor Internalization Using High-Throughput Technologies

 Materials
  • Cells of interest grown in 96-well plates (see Support Protocol 1)
  • Assay buffer (see recipe)
  • 4% (w/v) paraformaldehyde containing 15 µg/ml Hoechst 3334
  • Plates containing agonist or antagonist (see Support Protocol 2)
  • Multichannel pipettors (e.g., Biomek Fx, Multimek Multi-Channel Pipettor, and Biomek 2000 laboratory automated workstation; Beckman Coulter) and appropriate pipet tips
  • Plate washer (Elx405; Bio-Tek Instruments) with 96-well pipetting head
  • Arrayscan II imaging system (Cellomics)
  • Analysis software, such as Microsoft Excel (Microsoft) or GraFit version 4.0.13 (Erithacus Software)

Support Protocol 1: Preparation of Cells for Receptor Internalization Studies

 Materials
  • Cells grown to confluence in 175-cm2 (T175) flask
  • PBS Dulbecco's without Ca2+ and Mg2+ (Life Technologies)
  • Versene (Life Technologies)
  • Standard growth medium for cells being seeded
  • Assay buffer (see recipe)
  • Biocoat poly-l-lysine-coated black-walled plates (Becton Dickinson)
  • 50-ml centrifuge tubes (Nunc)
  • Benchtop centrifuge (e.g., CR4.22, Jouan)
  • Multi-well plates with or without appropriate coverslips or chambered coverglass slides for confocal microscopy
  • Additional reagents and equipment for counting cells

Support Protocol 2: Preparation of Ligand and Compound Plates for Receptor Internalization Studies

 Materials
  • Agonist of interest
  • Assay buffer (see recipe)
  • Agonist or antagonist to be tested
  • 96-well polypropylene round-bottom plates (Becton Dickinson)
  • Biomek 2000 laboratory automated workstation (Beckman Coulter) and appropriate pipet tips
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 12.6.1
    [3H]Apelin-13-apelin receptor complexes are rapidly internalized into HEK293 cells. HEK293 cells stably expressing the apelin receptor were treated with [3H]apelin (10 nM) for varying periods of time. The extent of internalization of specifically bound [3H]apelin was then measured. Data represent mean ± standard error of three experiments performed in triplicate.

    View Image
  • Figure 12.6.2
    HEK293 cells stably expressing receptor–green fluorescent protein (GFP) chimera before and after ligand addition. (A) HEK293 cells stably expressing the receptor-GFP chimera before addition of ATP to the cells. (B) The same field of cells 45 min after addition of 2.5 µM ATP at 37°C. After ligand addition, internalized receptors can be visualized as punctate fluorescence within the cells.

    View Image
  • Figure 12.6.3
    CHO cells stably expressing the Orexin 1 receptor following incubation with rhodamine-labeled Orexin A. Tetramethylrhodamine (Tamra)-labeled Orexin A (0.5 µM) was incubated with CHO cells expressing the Orexin 1 receptor for 45 min at 37°C. Cells were subsequently fixed and stained with Hoechst 33342 to identify nuclei (blue). Endocytosed ligand (red) can be seen around the perinuclear region.

    View Image
  • Figure 12.6.4
    Concentration-response curve to tetramethylrhodamine (Tamra)-labeled Orexin A (Tamra-Ox-A), using CHO cells that stably express the Orexin 1 receptor. Data were generated by the Cellomics Arrayscan II instrument. Analysis was carried out upon images similar to that in Figure 12.6.3. Two images per well were analyzed in triplicate for each concentration. The pEC50 (–log[EC50]) value generated in this manner was 8.8; the pEC50 value produced by Ca2+ assay using FLIPR technology was 8.3. Error bars represent the standard deviation.

    View Image
  • Figure 12.6.5
    Inhibition response curve using CHO cells stably expressing the Orexin 1 receptor, with Orexin A antagonist SB-334867 versus 40 nM tetramethylrhodamine (Tamra)-labeled Orexin A. Data were generated by the Cellomics Arrayscan II instrument. Image analysis was performed similar to that in Figure 12.6.3. Two images per well were analyzed in triplicate for each concentration. The pKB value generated in this manner was 7.34; the pKB value produced by Ca2+ assay using FLIPR technology was 7.27. Error bars are SEM.

    View Image

Literature Cited

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